Genetic Modification of in vitro Primary Cultured Human Corneal Endothelial Cells With Equine Infectious Anemia Virus

Abstract

Purpose: : Lentiviral vectors are powerful tools for long–term genetic modification of target cells. Equine infectious anemia virus (EIAV) is a non–primate lentiviral vector shown to transduce both dividing and nondividing cells at efficiencies comparable to HIV–based vectors in a variety of tissues. In a previous study, EIAV has been used to modify ex vivo human corneal endothelium. The present study was undertaken to assess the efficiency and toxicity of EIAV transduction of primary cultured human corneal endothelial cells (HCECs).

Methods: : A three–plasmid system and 293–T cells were used to produce EIAV particles pseudotyped with vesicular stomatitis virus envelope protein and encoding enhanced green fluorescent protein (eGFP) as a marker gene (Oxford Biomedica, Oxford, UK). Endothelial cells obtained from a 3 year old human cornea were dissociated from Descemet membrane, and cells were grown in organ culture medium containing OptiMEM–1, fetal bovine serum, epidermal growth factor, nerve growth factor, bovine pituitary extract, ascorbic acid, calcium chloride, chondroitin sulfate, gentamicin, and antimycotic solution. The cells were grown to 70–80% confluence. EIAV particles at 1.0 x 10⁸ TU/ml were incubated with the HCECs at 37^oC for 12 hours. Fresh medium was then added, and cells were examined by fluorescence microscopy. Cells expressing eGFP vector were collected via fluorescence activated cell sorting (FACS).

Results: : EIAV transduction resulted in eGFP expression in approximately 30–40% of primary corneal endothelial cells. eGFP expression was detected in 97–99% of human corneal endothelial cells after FACS sorting. Cell morphology, growth rate, and percent viability showed no detectable differences in transduced vs. non–transduced cells.

Conclusions: : EIAV transduction results in high efficiency gene transfer to primary human corneal endothelial cells which can be further optimized with FACS sorting. No toxic effects of this vector were observed at the particle concentration used in this study. Longer term assessment of marker gene expression and toxicity are underway. These results suggest the EIAV vector may be useful for cell– and gene–based therapies of corneal endothelial disorders.

Keywords: cornea: endothelium • gene transfer/gene therapy